Environmental Science and Pollution Research

, Volume 26, Issue 4, pp 4116–4129 | Cite as

Biochemical, molecular, and elemental profiling of Withania somnifera L. with response to zinc stress

  • Jyoti Ranjan RoutEmail author
  • Rout George Kerry
  • Debasna Panigrahi
  • Santi Lata Sahoo
  • Chinmay Pradhan
  • Shidharth Sankar Ram
  • Anindita Chakraborty
  • Mathummal Sudarshan
Research Article


Zn stress seriously induces various toxic responses in Withania somnifera L., when accumulated above the threshold level which was confirmed by investigating the responses of protein, expression of antioxidant enzymes, and elemental profiling on accumulation of Zn. Zn was supplemented in the form of ZnSO4 (0, 25, 50, 100, and 200 μM) through MS liquid medium and allowed to grow the in vitro germinated plants for 7 and 14 days. The study revealed that when the application of Zn increased, a significant reduction of growth characteristics was noticed with alterations of proteins (both disappearance and de novo synthesis). The activity of CAT, SOD, and GPX were increased up to certain concentrations and then declined, which confirmed through in-gel activity under different treatments. RT-PCR was conducted by taking three sets of genes from CAT (RsCat, Catalase1, Cat1) and SOD (SodCp, TaSOD1.2, MnSOD) and found that gene RsCat from CAT and MnSOD from SOD have shown maximum expression of desired genes under Zn stress, which indicate plant’s stress tolerance mechanisms. The proton-induced X-ray emission study confirmed an increasing order of uptake of Zn in plants by suppressing and expressing other elemental constituents which cause metal homeostasis. This study provides insights into molecular mechanisms associated with Zn causing toxicity to plants; however, cellular and subcellular studies are essential to explore molecule-molecule interaction during Zn stress in plants.


Antioxidant enzymes Ashwagandha Gene expression Phytotoxicity PIXE Zinc excess 





Murashige and Skoog


Reverse transcriptase polymerase chain reaction




Superoxide dismutase


Proton-induced X-ray emission


Reactive oxygen species


Sodium dodecyl sulfate polyacrylamide gel electrophoresis


Ethylenediamine-tetraacetic acid


Guaiacol peroxidase





The authors are grateful to the Director, Institute of Physics, Bhubaneswar, for providing PIXE facility to study the metal analysis.

Funding information

This study received financial support from UGC-DAE Consortium for Scientific Research, Kolkata, India (Grant No. UGC-DAE-CSR-KC/CRS/2009/TE-01/1539).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Aebi H (1983) In: Bergmeyer HU (ed) Methods of Enzymatic Analysis. Verlag Chemie, Weinheim, pp 673–684Google Scholar
  2. Appenroth KJ (2010) What are “heavy metals” in plant sciences? Acta Physiol Plant 32:615–619Google Scholar
  3. Ara N, Nakkanong K, Lv W, Yang J, Hu Z, Zhang M (2013) Antioxidant enzymatic activities and gene expression associated with heat tolerance in the stems and roots of two cucurbit species (“Cucurbita maxima” and “Cucurbita moschata”) and their interspecific inbred line “Maxchata”. Int J Mol Sci 14(12):24008–24028Google Scholar
  4. Arough YK, Sharifi RS, Sharifi RS (2016) Bio fertilizers and zinc effects on some physiological parameters of triticale under water-limitation condition. J Plant Interact 11(1):167–177Google Scholar
  5. Barker AV, Eaton TE (2015) Zinc. In: Barker AV, Pilbeam DJ (eds) Hand book of plant nutrition, Second edn. CRC Press, Florida, p 537Google Scholar
  6. Barua P, Gayen D, Lande NV, Chakraborty S, Chakraborty N (2017) Global proteomic profiling and identification of stress-responsive proteins using two-dimensional gel electrophoresis. Methods Mol Biol 1631:163–179Google Scholar
  7. Beauchamp C, Fridovich I (1971) Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal Biochem 44:276–287Google Scholar
  8. Bergmeyer HU (1974) In: Bergmeyer, H.U. (Eds.), Methods of enzymatic analysis. Academic Press, New York, London, pp. 673–677Google Scholar
  9. Bernhard R, Verkleij JAC, Nelissen HJM, Vink JPM (2005) Plant specific responses to zinc contamination in a semi-field lysimeter and on hydroponics. Environ Pollut 138:100–108Google Scholar
  10. Britto AJD, Raj TLS, Sutha M (2013) Molecular responses of groundnut (Arachis hypogea L.) to zinc stress. J Stress Physiol Biochem 9(3):152–158Google Scholar
  11. Ceylan S, Soya H, Budak B, Akdemir H, Esetlili BC (2009) Effect of zinc on yield and some related traits of alfalfa. Turk J Field Crops 14(2):136–143Google Scholar
  12. Chaoui A, Mazhoudi S, Ghorbal MH, Ferjani EE (1997) Cadmium and zinc induction of lipid peroxidation and effects on antioxidant enzyme activities in bean (Phaseolus vulgaris L.). Plant Sci 127:139–147Google Scholar
  13. Clemens S (2001) Molecular mechanisms of plant metal tolerance and homeostatis. Planta 212(4):475–486Google Scholar
  14. Cobbett CS (2000) Phytochelatins and their roles in heavy metal detoxification. Plant Physiol 123(3):825–832Google Scholar
  15. Cook D, Fowler S, Fiehn O, Thomashow MF (2004) A prominent role for the CBF cold response pathway in configuring the low-temperature metabolome of Arabidopsis. Proc Natl Acad Sci U S A 101:15243–15248Google Scholar
  16. Cui Y, Zhao N (2011) Oxidative stress and change in plant metabolism of maize (Zea mays L.) growing in contaminated soil with elemental sulfur and toxic effect of zinc. Plant Soil Environ 57(1):34–39Google Scholar
  17. Damerval C, Vienne P, Zivy M, Thiellement H (1986) Technical improvement in two-dimensional electrophoresis increase the level of genetic variation detected in wheat seedling proteins. Electrophoresis 7:52–54Google Scholar
  18. Das K, Samanta L, Chainy GBN (2000) A modified spectrophotometric assay of superoxide dismutase using nitrite formation by superoxide radicals. Indian J Biochem Biophys 37:201–204Google Scholar
  19. Emamverdian A, Ding Y, Mokhberdoran F, Xie Y (2015) Heavy metal stress and some mechanisms of plant defense response. Sci World J 2015:1–18Google Scholar
  20. Ferreira RR, Fornazier RF, Vitoria AP, Lea PJ, Azevedo RA (2002) Changes in antioxidant enzyme activities in soybean under cadmium stress. J Plant Nutr 25:327–342Google Scholar
  21. Fidalgo F, Azenha M, Silva AF, Sousa A, Santiago A, Ferraz P, Teixeira J (2013) Copper-induced stress in Solanum nigrum L. and antioxidant defense system responses. Food Energy Secu 2(1):70–80Google Scholar
  22. Godina RGC, Pournavab RF, Mendoza AB (2016) Effect of selenium on elemental concentration and antioxidant enzymatic activity of tomato plants. J Agric Sci Technol 18:233–244Google Scholar
  23. Halliwell B, Gutteridge JMC (2007) Free radicals in biology and medicine, Fourth edn. Oxford University Press, New YorkGoogle Scholar
  24. Hamill DE, Brewbaker JL (1969) Isoenzyme polymorphism in flowering plants. IV. The peroxidase isoenzymes of maize (Zea mays L.). Physiol Plant 22:945–958Google Scholar
  25. Hasan MK, Cheng Y, Kanwar MK, Chu XY, Ahammed GJ, Qi ZY (2017) Responses of plant proteins to heavy metal stress-a review. Front Plant Sci 8:1–16Google Scholar
  26. He J, Wang Y, Ding H, Ge C (2016) Epibrassinolide confers zinc stress tolerance by regulating antioxidant enzyme responses, osmolytes, and hormonal balance in Solanum melongena seedlings. Braz J Plant Physiol 39(1):295–303Google Scholar
  27. Hu Z, Wenjiao Z (2015) Effects of zinc stress on growth and antioxidant enzyme responses of Kandelia obovata seedlings. Toxicol Environ Chem 97(9):1190–1201Google Scholar
  28. Hussain I, Ashraf MA, Rasheed R, Saeed F (2017) Cadmium-induced perturbations in growth, oxidative defense system, catalase gene expression and fruit quality in tomato. Int J Agric Biol 19:61–68Google Scholar
  29. Israr M, Jewell A, Kumar D, Sahi SV (2011) Interactive effects of lead, copper, nickel and zinc on growth, metal uptake and antioxidative metabolism of Sesbania drummondii. J Hazard Mater 186(2-3):1520–1526Google Scholar
  30. Jahantigh O, Najafi F, Badi HN, Khavari-Nejad RA, Sanjarian F (2016) Changes in antioxidant enzymes activities and proline, total phenol and anthocyanine contents in Hyssopus officinalis L. plants under salt stress. Acta Biol Hung 67(2):195–204Google Scholar
  31. Jayasri MA, Suthindhiran K (2017) Effect of zinc and lead on the physiological and biochemical properties of aquatic plant Lemna minor: its potential role in phytoremediation. Appl Water Sci 7(3):1247–1253Google Scholar
  32. Jibril SA, Hassan SA, Ishak CF, Wahab PEM (2017) Cadmium toxicity affects phytochemicals and nutrient elements composition of lettuce (Lactuca sativa L.). Adv Agric 2017:1–7Google Scholar
  33. Kerry RG, Mahapatra GP, Patra S, Sahoo SL, Pradhan C, Padhi BK, Rout JR (2018) Proteomic and genomic responses of plants to nutritional stress. Biometals 31:161–187Google Scholar
  34. Kosesakal T, Unal M (2012) Effects of zinc toxicity on seed germination and plant growth in tomato (Lycopersicon esculentum Mill.). Fresenius Environ Bull 21(2):315–321Google Scholar
  35. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685Google Scholar
  36. Leng X, Jia H, Sun X, Shangguan L, Mu Q, Wang B, Fang J (2015) Comparative transcriptome analysis of grapevine in response to copper stress. Sci Rep 5:17749Google Scholar
  37. Leskova A, Giehl RFH, Hartmann A, Fargasova A, Wirena N (2017) Heavy metals induce iron deficiency responses at different hierarchic and regulatory levels. Plant Physiol 174:1648–1668Google Scholar
  38. Li H, Luo H (2012) Antioxidant enzyme activity and gene expression in response to lead stress in Perennial ryegrass. J Amer Soc Hort Sci 137(2):80–85Google Scholar
  39. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275Google Scholar
  40. Luo ZB, He XJ, Chen L, Tang L, Gao S, Chen F (2010) Effects of zinc on growth and antioxidant responses in Jatropha curcas seedlings. Int J Agric Biol 12:119–124Google Scholar
  41. Manivasagaperumal R, Balamurugan S, Thiyagarajan G, Sekar J (2011) Effect of zinc on germination, seedling growth and biochemical content of cluster bean (Cyamopsis tetragonoloba (L.) Taub). Curr Bot 2(5):11–15Google Scholar
  42. Matraszek R, Hawrylak-Nowak B, Chwil S, Chwil M (2016) Interaction between cadmium stress and sulphur nutrition level on macronutrient status of Sinapis alba L. Water Air Soil Pollut 227:355Google Scholar
  43. Michael PI, Krishnaswamy M (2011) The effect of zinc stress combined with high irradiance stress on membrane damage and antioxidative response in bean seedlings. Environ Exp Bot 74:171–177Google Scholar
  44. Mishra S, Srivastava S, Tripathi RD, Kumar R, Seth CS, Gupta DK (2006) Lead detoxification by coontail (Ceratophyllum demersum L.) involves induction of phytochelatins and antioxidant system in response to its accumulation. Chemosphere 65(6):1027–1039Google Scholar
  45. Mukhopadhyay M, Das A, Subba P, Bantawa P, Sarkar B, Ghosh P, Mondal TK (2013) Structural, physiological, and biochemical profiling of tea plantlets under zinc stress. Biol Plant 57(3):474–480Google Scholar
  46. Olafisoye OB, Ojelade OD, Osibote OA (2017) Trace elements and antioxidants in some medicinal plants. Res Rev Biosci 11(3):111Google Scholar
  47. Oliva SR, Mingorance MD, Valdes B, Leidi EO (2010) Uptake, localization and physiological changes in response to copper excess in Erica andevalensis. Plant Soil 328:441–420Google Scholar
  48. Olteanu Z, Truta E, Oprica L, Zamfirache MM, Rosu CM, Vochita G (2013) Copper-induced changes in antioxidative response and soluble protein level in Triticumae stivum cv. beti seedlings. Rom Agric Res 30:1–8Google Scholar
  49. Olteanu Z, Oprica L, Truta E, Lobiuc A, Zamfirache MM (2014) Effects induced by zinc on some antioxidative enzyme activities and on soluble protein content in young plantlets of barley. An Stiint Univ Al I Cuza din II a 15(2):23–30Google Scholar
  50. Ong GH, Yap CK, Maziah M, Tan SG (2013) Synergistic and antagonistic effects of zinc bioaccumulation with lead and antioxidant activities in Centella asiatica. Sains Malays 42(11):1549–1555Google Scholar
  51. Panda SK, Matsumoto H (2010) Changes in antioxidant gene expression and induction of oxidative stress in pea (Pisum sativum L.) under Al stress. Biometals 23(4):753–762Google Scholar
  52. Radic S, Babic M, Skobic D, Roje V, Pevalek-Kozlina B (2010) Ecotoxicological effects of aluminum and zinc on growth and antioxidants in Lemna minor L. Ecotoxicol Environ Saf 73(3):336–342Google Scholar
  53. Rahdari P, Hoseini SM, Movafegh S (2013) Alteration in metabolic process of Glysine max L. fellowing "Zn" rate changes. Intl J Agron Plant Prod 4(3):589–594Google Scholar
  54. Rastgoo L, Alemzadeh A (2011) Biochemical responses of Gouan (Aeluropus littoralis) to heavy metals stress. Aust J Crop Sci 5(4):375–383Google Scholar
  55. Rengel Z, Graham RD (1996) Uptake of zinc from chelate-buffered nutrient solutions by wheat genotypes differing in zinc efficiency. J Exp Bot 47(295):217–226Google Scholar
  56. Rout JR, Sahoo SL (2013) Antioxidant enzyme gene expression in response to copper stress in Withania somnifera L. Plant Growth Regul 71(1):95–99Google Scholar
  57. Rout JR, Ram SS, Das R, Chakraborty A, Sudarshan M, Sahoo SL (2013) Copper-stress induced alterations in protein profile and antioxidant enzymes activities in the in vitro grown Withania somnifera L. Physiol Mol Biol Plants 19:353–361Google Scholar
  58. Rout JR, Behera S, Keshari N, Ram SS, Bhar S, Chakraborty A, Sudarshan M, Sahoo SL (2015) Effect of iron stress on Withania somnifera L.: antioxidant enzyme response and nutrient elemental uptake of in vitro grown plants. Ecotoxicology 24(2):401–413Google Scholar
  59. Rout JR, Sahoo SL, Das R, Ram SS, Chakraborty A, Sudarshan M (2017) Changes in antioxidant enzyme activities and elemental profiling of Abutilon indicum L. subjected to copper stress. Proc Natl Acad Sci India Sect B Biol Sci 87(4):1469–1478Google Scholar
  60. Roy SK, Cho SW, Kwon SJ, Kamal AH, Kim SW, Oh MW, Lee MS, Chung KY, Xin Z, Woo SH (2016) Morpho-physiological and proteome level responses to cadmium stress in Sorghum. PLoS One 11(2):1–27Google Scholar
  61. Shah K, Dubey RS (1998) Cadmium elevates the protein level and alters the activity of proteolytic enzymes in germinating rice seeds. Acta Physiol Plant 20(2):189–196Google Scholar
  62. Shah K, Kumar RG, Verma S, Dubey RS (2001) Effect of cadmium on lipid peroxidation, superoxide anion generation and activities of antioxidant enzymes in growing rice seedlings. Plant Sci 161(6):1135–1141Google Scholar
  63. Shanker AK (2008) Mode of action and toxicity of trace elements. In: Prasad MNV (ed) Trace elements: Nutritional benefits, environmental contamination and health implications. John Wiley & Sons Inc., New York, pp 525–555Google Scholar
  64. Singh PK, Tewari RK (2003) Cadmium toxicity induced changes in plant water relations and oxidative metabolism of Brassica juncea L. plants. J Environ Biol 24(1):107–112Google Scholar
  65. Singh S, Parihar p SR, Singh VP, Prasad SM (2016) Heavy metal tolerance in plants: role of transcriptomics, proteomics, metabolomics, and ionomics. Front Plant Sci 6:1147Google Scholar
  66. Stefanic PP, Sikic S, Cvjetko C, Balen B (2012) Cadmium and zinc induced similar changes in protein and glycoprotein patterns in tobacco (Nicotiana tabacum L.) seedlings and plants. Arh Hig Rada Toksikol 63:321–335Google Scholar
  67. Stoyanova Z, Doncheva S (2002) The effect of zinc supply and succinate treatment on plant growth and mineral uptake in pea plant. Braz J Plant Physiol 14(2):111–116Google Scholar
  68. Sun BY, Kan SH, Zhang YZ, Deng SH, Wu J, Yuan H, Qi H, Yang G, Li L, Zhang XH, Xiao H, Wang YJ, Peng H, Li YW (2010) Certain antioxidant enzymes and lipid peroxidation of radish (Raphanus sativus L.) as early warning biomarkers of soil copper exposure. J Hazard Mater 183(1-3):833–838Google Scholar
  69. Switzer RC (1979) A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels. Anal Biochem 98:231–237Google Scholar
  70. Wang C, Zhang SH, Wang PF, Hou J, Zhang WJ, Li W, Lin ZP (2009) The effect of excess Zn on mineral nutrition and antioxidative response in rapeseed seedlings. Chemosphere 75:468–1476Google Scholar
  71. Woodbury W, Spencer AK, Stahman MA (1971) An improved procedure using ferricyanide for detecting catalase isozymes. Anal Biochem 44:301–305Google Scholar
  72. Wu T, Lee T (2008) Regulation of activity and gene expression of antioxidant enzymes in Ulva fasciata Delile (Ulvales, Chlorophyta) in response to excess copper. Phycologia 47(4):346–360Google Scholar
  73. Yadav SK (2010) Heavy metals toxicity in plants: an overview on the role of glutathione and phytochelatins in heavy metal stress tolerance of plants. S Afr J Bot 76:167–179Google Scholar
  74. Youssef MM, Azooz MM (2013) Biochemical studies on the effects of zinc and lead on oxidative stress, antioxidant enzymes and lipid peroxidation in okra (Hibiscus esculentus cv. Hassawi). Sci International 1(3):29–38Google Scholar
  75. Yu R, Tang Y, Liu C, Du X, Miao C, Shi G (2017) Comparative transcriptomic analysis reveals the roles of ROS scavenging genes in response to cadmium in two pakchoi cultivars. Sci Rep 7:9217Google Scholar
  76. Zhang Y, Han X, Chen S, Zheng L, He X, Liu M, Qiao G, Wang Y, Zhuob R (2017) Selection of suitable reference genes for quantitative real-time PCR gene expression analysis in Salix matsudana under different abiotic stresses. Sci Rep 7:40290Google Scholar
  77. Zhao H, Wu L, Chai T, Zhang Y, Tan J, Ma S (2012) The effects of copper, manganese and zinc on plant growth and elemental accumulation in the manganese-hyperaccumulator Phytolacca americana. J Plant Physiol 169(13):1243–1252Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Jyoti Ranjan Rout
    • 1
    • 4
    Email author
  • Rout George Kerry
    • 2
  • Debasna Panigrahi
    • 3
  • Santi Lata Sahoo
    • 4
  • Chinmay Pradhan
    • 4
  • Shidharth Sankar Ram
    • 5
  • Anindita Chakraborty
    • 5
  • Mathummal Sudarshan
    • 5
  1. 1.School of Biological SciencesAIPH UniversityBhubaneswarIndia
  2. 2.Post Graduate Department of BiotechnologyUtkal UniversityBhubaneswarIndia
  3. 3.Department of Life ScienceNational Institute of Technology RourkelaRourkelaIndia
  4. 4.Biochemistry and Molecular Biology Laboratory, Post Graduate Department of BotanyUtkal UniversityBhubaneswarIndia
  5. 5.UGC-DAE Consortium of Scientific ResearchKolkata CentreKolkataIndia

Personalised recommendations